Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of chronic morbidity and mortality in the United States and is projected to become the third leading cause of death worldwide by 2020 (Mannino & Braman 2007, Proceedings of the American Thoracic Society, vol. 4, pp. 502-506; Rabe et al. 2007, Am J Respir. Crit Care Med., vol. 176, no. 6, pp. 532-555). Cigarette smoking is recognized as the most important environmental causative factor for COPD (Mannino & Braman 2007; Marsh et al. 2006, European Respiratory Journal, vol. 28, pp. 883-886; Rabe et al. 2007). It is estimated that up to 50% of smokers may eventually develop COPD, as defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) spirometric criteria (Lokke et al. 2006, Thorax, vol. 61, pp. 935-939; Lundbäck et al. 2003, Respiratory Medicine, vol. 97, pp. 115-122; Mannino & Braman 2007). COPD is characterized by incompletely reversible airflow limitation that results from small airway disease (obstructive bronchiolitis) and parenchymal destruction (emphysema). These pathologic changes are the result of an abnormal inflammatory response to long-term exposure to noxious gases or particles, with structural changes due to repeated injury and repair (Rabe et al. 2007). The mechanisms of the enhanced inflammation that characterizes COPD involve both innate and adaptive immunity in response initially to inhalation of particles and gases (MacNee 2001, Journal of Pharmacology, vol. 429, pp. 195-207). Several studies have demonstrated differences in markers of inflammation and immune response, such as a correlation between the number of CD8 cytotoxic T lymphocytes and the degree of airflow limitation in COPD (Curtis, Freeman, & Hogg 2007, Proceedings of the American Thoracic Society, vol. 4, no. 7, pp. 512-521). The response to oxidative stress is considered an important factor in the pathogenesis of COPD (MacNee 2005, Proceedings of the American Thoracic Society, vol. 2, no. 1, pp. 50-60), while protease-antiprotease imbalance is thought to be associated with emphysema (Baraldo et al. 2007, Chest, vol. 132, no. 6, pp. 1733-1740). However, while inflammation and other factors are clearly involved in the molecular pathogenesis of COPD, the precise etiological mechanisms remain to be fully characterized.
Recent advances in laboratory technologies have enabled “omics” investigations that allow researchers to interrogate diseases on an unprecedented scale (Evans 2000, Nature Biotechnology, vol. 18, no. 2, p. 127). A rapidly developing discipline in this area is metabolomics, which is the study of all measurable metabolites in a given biological sample (Kaddurah-Daouk, Kristal, & Weinshilboum 2008, Annu. Rev. Pharmacol. Toxicol., vol. 48, pp. 653-683). Metabolomics studies typically employ either mass spectrometry or nuclear magnetic resonance (NMR) to assay the biochemical components of a tissue or biofluid. Proton (1H) NMR metabolomics has already shown considerable potential as a diagnostic tool for Alzheimer's disease (Tukiainen et al. 2008, Biochem. Biophys. Res Commun., vol. 375, no. 3, pp. 356-361), diabetes and related disorders (Makinen et al. 2006, MAGMA., vol. 19, no. 6, pp. 281-296; Makinen et al. 2008, Mol Syst. Biol., vol. 4, p. 167) and inborn errors of lipid metabolism (Oostendorp et al. 2006, Clin. Chem., vol. 52, no. 7, pp. 1395-1405). Furthermore, 1H NMR metabolomics can facilitate the discovery of novel candidate biomarkers of disease risk, as demonstrated by Holmes et al. (2008, Nature, vol. 453, no. 7193, pp. 396-400) in a recent population study of blood pressure. Finally, considering pulmonary studies specifically, de Laurentiis et al. (2008, Eur, Respir. J, vol. 32, no. 5, pp. 1175-1183) found that NMR-based metabolic profiles of exhaled breath condensate enabled discrimination between individuals with and without COPD.
An individual's overall ‘metabolic phenotype’ reflects an individual's metabolic characteristics and is determined by the interaction of an individual's genetic makeup and his/her adaptive response to the environment. As described by Bemini et al. (J. Proteome Res., 2009, 8 (9), pp 4264-4271), any “differences between individual phenotypes are due both to differences in genotype and exposure to different environmental factors . . . [I]ndividual metabolic phenotype can also be considered a metagenomic entity that is strongly affected by both gut microbiome and host metabolic phenotype, the latter defined by both genetic and environmental contribution.” Bernini et al. Thus, differences in cellular processes due to exposure to harmful substances are reflected as differences in metabolic phenotypes in subjects having a lung disease such as, for example, COPD as compared to normal (healthy) subjects.
The biochemical composition resulting from an individual's metabolic processes is ultimately reflected in the extracellular tissue fluid and consequently in an individual's biofluids such as blood and urine. Consequently, abnormal cellular metabolic processes can effect compositions of blood and urine. As such, these fluids may provide diagnostic and prognostic windows onto an individual's “phenotypic state”. For example, in subjects having the disorder phenylketonuria (deficiency of a phenylalanine converting enzyme), increased concentration of phenylpyruvic acid, phenyllactic acid and phenylacetic acid are present in urine from such subjects as compared to urine of normal subjects. See, e.g., Text Book of Biochemistry with Clinical Correlations, 4th Edition, 1997, edited by T. M. Devin, published by Wiley-Liss. In such cases, urinary biochemical analysis can diagnose or aid in the diagnosis of those subjects having the enzyme deficiency.
In lung disorder such as, for example, COPD, factors influencing lung function and its decline include environmental and biological effects (Feenstra et al. 2001, Am J Respir. Crit Care Med., vol. 164, no. 4, pp. 590-596; Hoidal 2001, Eur. Respir. J, vol. 18, no. 5, pp. 741-743; Sandford & Silverman 2002, Thorax, vol. 57, no. 8, pp. 736-741). Although factors associated with lung function decline in middle-aged and older adults have been identified in cross-sectional studies (Enright et al. 1994; Kerstjens, Brand, and Postma 1996), predictions based on such studies may not adequately predict longitudinal changes within individuals (Knudson et al. 1983, American Review of Respiratory Disease, vol. 127, pp. 725-734; Griffith et al. 2001, American Journal of Respiratory and Critical Care Medicine, vol. 163, pp. 61-68).
Prior diagnostic methods of COPD and other lung diseases employ diagnostic tests which rely on the presumed correlation of functional measures of decreased lung function with lung disease. Spirometry, which is the most commonly performed lung function test, is a simple breathing test that measures the quantity of air that a subject can expel and the speed with which the air is expelled. While lung function tests can provide a general assessment of the functional status of a subject's lungs, it does not distinguish between the different types of lung diseases that may be present. Certain diseases such as asthma for example cannot be confirmed based on functional tests alone. In addition, it is only when a change in lung function exists can such tests assist in the diagnosis of lung disease. Functional diagnostic methods do not predict the onset or progression of the disease.
In contrast to diagnostics based on lung function, proton nuclear magnetic resonance spectroscopy, as described herein, can be used to identify and quantify metabolites associated with decreased lung function without functional assessment.